The glucocorticoid receptor (GR) is associated through its hormone binding domain (HBD) with the abundant and ubiquitous protein chaperone hsp90. Binding to hsp90 is required for the GR to have steroid binding activity and for hormonal control of receptor function. GR- hsp90 heterocomplexes are assembled by a multi-protein chaperone system in reticulocyte lysate that we have reconstituted from five purified components during the past seven years. This assembly system forms complexes between hsp90 and a variety of transcription factors and protein kinases involved in signal transduction, cell cycle control and oncogenesis. The components of the assembly system from pants and animals compliment each other when placed in the heterologous purified system, suggesting that this hsp90-based protein folding system serves a basic, conserved function in all eukaryotic cells. The five protein assembly machinery consists of the essential chaperones hsp90 and hsp70 and the non-essential cp-chaperones hsp40, Hop and p23. Recently, we have developed a two-step system of GR-hsp90 heterocomplex assembly in which immobilized GR is incubated with purified hsp70, washed free of unbound protein, and then incubated with the other chaperones. The first step in the assembly is the ATP- dependent and hsp40-dependent formation of a GR-hsp70 complex that is now 'primed' to bind hsp90 in a second ATP-dependent assembly step. Steroid binding activity is generated during the second incubation with gsp90. Specific aims 1-5 of this proposal focus on the mechanism of heterocomplex assembly by determining the stochiometry of gsp70 to GR in the 'primed' GR.hsp70 complex, the site of hsp70binding to the GR HBD, the nucleotide binding state of hsp70 hsp90 at each step of assembly, the potential for GR-bound hsp70 (and possibly hsp90) to ratchet back and forth between ATP-dependent and ADP-dependent conformations during assembly, and the role of hsp90 phosphorylation in its assembly activity. Several lines of evidence support the proposal that the hsp90/hsp70- based chaperone machinery acts to open the hydrophobic ligand binding cleft in the GR HBD to access by hormone. Specific aim 6 is designed to further test this model and to see if it can be applied to non-receptor proteins in the opening of hydrophobic clefts to access by other ligands.